Donor UNC-93 Homolog B1 Genetic Polymorphism Predicts Survival Outcomes After Unrelated Bone Marrow Transplantation

Genes & Immunity (2021) 22:35–43 https://doi.org/10.1038/s41435-021-00122-y

ARTICLE

Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation

1,2 1 1,2 1,2 1,2 Kaori Uchino ● Lam Vu Quang ● Shohei Mizuno ● Tomohiro Horio ● Hidesuke Yamamoto ● 1,2 2 3 4 5 Ichiro Hanamura ● Yoshihisa Kodera ● J. Luis Espinoza ● Makoto Onizuka ● Koichi Kashiwase ● 6,7,8 9 10 11 12 13 Yasuo Morishima ● Takahiro Fukuda ● Noriko Doki ● Koichi Miyamura ● Takehiko Mori ● Eriko Morishita ● 14 1,2 Shinji Nakao ● Akiyoshi Takami

Received: 28 August 2020 / Revised: 19 January 2021 / Accepted: 27 January 2021 / Published online: 24 February 2021 © The Author(s), under exclusive licence to Springer Nature Limited 2021

Abstract UNC-93 homolog B1 (UNC93B1) is a key regulator of toll-like receptors (TLRs), pattern recognition receptors that sense invading pathogens and manage the innate immune response and deliver them from the endoplasmic reticulum to their respective endosomal signaling compartments. Several types of TLRs are known to contribute to the inﬂammatory process after allogeneic hematopoietic stem cell transplantation (SCT), so UNC93B1 might play integral roles there. We investigated ﬂ

1234567890();,: 1234567890();,: the in uence of the UNC93B1 single-nucleotide polymorphism (SNP) rs308328 (T>C) on transplant outcomes in a cohort of 237 patients undergoing unrelated HLA-matched bone marrow transplantation (BMT) for hematologic malignancies through the Japan Marrow Donor Program. The donor UNC93B1 C/C genotype was associated with a better 3-year overall survival than the donor UNC93B1 C/T or T/T genotype. An analysis of the UNC93B1 rs308328 genotype may therefore be useful for selecting the donor, estimating the prognosis, and creating therapeutic strategies after allogeneic SCT.

Introduction such as severe infection, organ damage, and graft-versus- host disease (GVHD), remain obstacles to overcome [1]. Allogeneic hematopoietic stem cell transplantation (SCT) is Recently, increasing evidence has suggested that non-HLA expected to cure hematologic malignancies. However, life- genetic polymorphisms signiﬁcantly inﬂuence outcomes threatening complications associated with allogeneic SCT, after allogeneic SCT [2–13].

* Akiyoshi Takami 8 Department of Hematology and Oncology, Nakagami Hospital, [email protected] Okinawa, Japan 1 Division of Hematology, Department of Internal Medicine, Aichi 9 Hematopoietic Stem Cell Transplantation Unit, National Cancer Medical University School of Medicine, Nagakute, Japan Center Hospital, Tokyo, Japan 2 Hematopoietic Cell Transplantation Center, Aichi Medical 10 Hematology Division, Tokyo Metropolitan Cancer and Infectious University Hospital, Nagakute, Japan Diseases Center Komagome Hospital, Tokyo, Japan 3 Division of Rehabilitation Science, Faculty of Medicine, Institute 11 Department of Hematology, Japanese Red Cross Nagoya First of Medical, Pharmaceutical and Health Sciences, Kanazawa Hospital, Nagoya, Japan University, Kanazawa, Japan 12 Division of Hematology, Department of Medicine, Keio 4 Department of Hematology and Oncology, Tokai University University School of Medicine, Tokyo, Japan School of Medicine, Isehara, Japan 13 Department of Clinical Laboratory Sciences, Faculty of Medicine, 5 Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Institute of Medical, Pharmaceutical and Health Sciences, Tokyo, Japan Kanazawa University, Kanazawa, Japan 6 Department of Promotion for Blood and Marrow Transplantation, 14 Department of Hematology, Faculty of Medicine, Institute of Aichi Medical University School of Medicine, Nagakute, Japan Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan 7 Central Japan Cord Blood Bank, Seto, Japan 36 K. Uchino et al.

Toll-like receptors (TLRs) are the most important family Table 1 Patient, donor, and healthy volunteers characteristics. of receptors in the early to middle stages of infectious Variable Value immunity, cooperatively recognizing patterns present in microorganisms and augmenting the synthesis of inﬂamma- Number of cases 237 – tory mediators [14–17]. Previous studies [18–21]havesug- Patient age, years, median (range) 48 (1 67) – gested that the TLR -signaling pathway plays important roles Donor age, years, median (range) 34 (20 66) in the anti-microbial immunity and GVHD after allogeneic Healthy volunteers age, years, median (range) 30 (22–54) SCT. TLR genes have several functional single-nucleotide Year of HSCT, median (range) 2008 (2006–2009) polymorphisms (SNPs), which have been shown to be Patient UNC93B1 genotype, n (%) associated with the survival outcomes after allogeneic SCT in C/C 32 (14) our recent reports [22, 23]. TLRs are present not only on the C/T 95 (40) cell surface but also inside the cell, and such intracellular T/T 110 (46) TLRs are translocated from the ER to endolysosomes, which Donor UNC93B1 genotype, n (%) are critically regulated by UNC-93 homolog B1 (UNC93B1), C/C 33 (14) an ER protein with 12 membrane-spanning domains [24]. C/T 96 (41) UNC93B1 is encoded by the UNC93B1 gene on chromo- T/T 108 (46) some 11q13 and has one important SNP rs308328 (T>C) in Healthy volunteers UNC93B1 genotype, n (%) an intronic region that is functional, and the major allele (T) C/C 4 (17) has been reported to be associated with a lower UNC93B1 C/T 10 (43) expression than T allele-negative individuals [25]. T/T 21 (44) Given the above, we hypothesized that the SNP of Patient sex, n (%) UNC93B1 rs308328 may be associated with clinical out- Male 137 (58) comes after allogeneic SCT through affecting the function Female 100 (42) of UNC93B1. Donor sex, n (%) Male 157 (66) Results Female 80 (34) Patient/donor sex match, n (%) Frequencies of UNC93B1 genotypes Sex-matched 131 (55) Female/male 63 (27) The rs308328 (T>C) polymorphism in the UNC93B1 gene Male/female 43 (18) was genotyped in 237 patients with hematologic malig- Disease, n (%) nancies and their unrelated donors in this cohort (Table 1). AML 115 (49) The frequencies of C/C, C/T, and T/T in the rs308328 ALL 46 (19) (T>C) polymorphism were 14%, 40%, and 46% in the MDS 38 (16) patients and 14%, 41%, and 46% in the donors (P = 0.98), ML 24 (10) respectively. CML 10 (4.2) The prevalence of the UNC93B1 rs308328 C/C genotype MPD 3 (1.3) in the healthy Japanese population was 17% in according to Multiple myeloma 1 (0.42) a database of 1000 Genomes (https://www.internationa Myeloid malignancies 166 (70) lgenome.org/home). Lymphoid malignancies 71 (30) The UNC93B1 rs308328 genotype was also screened in Disease stage, n (%) 35 healthy Japanese volunteers in this study. The frequency High risk 119 (50) of C/C in these volunteers was 11% (Table 1). The allele Standard risk 118 (50) frequencies of this polymorphism in healthy controls, ABO matching, n (%) ﬁ donors and patients did not differ to a statistically signi cant ABO-matched 141 (60) = extent (P 0.13). Major mismatch 40 (17) Minor mismatch 43 (18) Transplant outcomes according to the UNC93B1 Bidirectional 13 (4.2) rs308328 genotypes Conditioning regimen, n (%) Myeloblastic 178 (75) Univariate analyses (Table 2) showed that the donor UNC93B1 rs308328 C/C genotype was associated with a Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow. . . 37

Table 1 (continued) P Variable Value

Reduced intensity 59 (25) Pretransplantation CMV serostatus, n (%) CMV-positive recipient 181 (76) Chronic GVHD, % Missing 16 (6.8)

PS, n (%) P PS0–1 224 (95) PS2–4 13 (5.5) TNC, ×108/kg, median (range) 2.7 (0.54–6.3) HSCT hematopoietic stem cell transplantation, AML acute myeloid IV acute GVHD, % leukemia, ALL acute lymphoblastic leukemia, MDS ML malignant – lymphoma, CML chronic myeloid leukemia, MPN myeloproliferative graft-versus-host disease. neoplasm, CMV cytomegalovirus, PS performance status, TNC total number of nucleated cells harvested. Grades III GVHD P better 3-year overall survival (OS) than the donor UNC93B1 rs308328 C/T or T/T genotype (77% vs. 58%; P = 0.04; Fig. 1A). Three years was set as the study time point according to the median follow-up period among the – survivors (753 days; range, 6 1918 days). The donor IV acute GVHD, % – UNC93B1 rs308328 C/C genotype also exhibited a trend Grades II toward a lower 3-year transplant-related mortality (TRM; transplant-related mortality, 13% vs. 27%, P = 0.06; Fig. 1C) than other genotypes but TRM showed no marked reduction in the 3-year progression-free P survival (PFS; 69% vs. 56%, P = 0.16; Fig. 1B) and 3-year relapse rate (18% vs. 18%, P = 0.72; Fig. 1D). The recipient UNC93B1 rs308328 genotype did not significantly influence the transplant outcomes in this study (Table 2). After adjusting for clinical factors in the multivariate 3-Year relapse, % model (Table 3), the donor UNC93B1 rs308328 C/C gen- P otype remained associated with a better 3-year OS than progression-free survival, other genotypes (hazard ratio [HR], 0.37; 95% confidence PFS interval [CI], 0.16–0.88; P = 0.03). When the main causes of death were analyzed according to the UNC93B1 rs308328 genotype, the donor UNC93B1 3-Year TRM, % rs308328 C/C genotype showed half the incidence of P death attributed to infection compared with other genotypes overall survival, fi (Fig. 2), although there were no signi cant differences. OS

Discussion 3-Year PFS, %

The present study showed that the donor UNC93B1 P rs308328 C/C genotype, which is presumed to have greater inducibility of UNC93B1 than other genotypes [25], was associated with signiﬁcantly better survival outcomes than other genotypes in patients with hematological malig- rs308328 genotype nancies receiving HLA fully matched unrelated bone mar- rs308328 genotype Univariate analysis of the association between UNC93B1 polymorphism and post HSCT outcomes. UNC93B1 row transplantation (BMT). hematopoietic stem cell transplantation, The mechanisms through which the donor UNC93B1 UNC93B1 C/C (32)C/T (95)T/T (110)C/C (32) 51C/T or T/T (205) 64 60 62 51 1.00 1.00 0.38 59 51 60 59 51 1.00 1.00 0.37 27 24 27 25 27 1.00 1.00 0.91 14 16 15 23 23 0.52 0.81 0.24 33 37 35 33 33 1.00 1.00 0.88 7.8 14 11 8.6 8.6 0.98 0.98 0.72 27 26 27 45 0.22 0.22 0.06 45 ﬁ C/C (33)C/T (96)T/T (108)C/C (33) 77C/T or T/T (204) 48 47 58 77 0.08 0.14 0.04 50 69 59 54 69 0.51 0.51 0.16 34 21 13 27 13 0.11 0.25 0.06 16 20 18 18 18 1.00 1.00 0.79 38 33 35 30 30 1.00 1.00 0.61 14 9.4 11 5.2 5.2 0.78 0.90 0.32 25 35 30 23 0.86 0.33 0.36 23 Table 2 Variable 3-Year OS, % HSCT Recipient rs308328 C/C genotype exerts its bene cial effects remain Donor 38 K. Uchino et al.

Fig. 1 Survival outcomes after transplantation according to the transplantation according to the donor UNC93B1 rs308328 genotype. donor UNC93B1 rs308328 genotype. The Kaplan–Meier analysis of The solid lines represent the donor C/C genotype, and the dashed lines the overall survival rates (A), the progression-free survival rates (B), represent the donor T/T or C/T genotype. the transplant-related mortality rates (C), and relapse rates (D) after to be determined. One plausible explanation is that patients (IL)-12p40 (T. gondii, T. cruzi). In another experimental transplanted from donors with the UNC93B1 C/C genotype study [32], other UNC93B1 mutant mice with a 54-amino may have been less susceptible to infection than those acid deletion in exon 4, which are also deficient in func- without the UNC93B1 C/C genotype, considering that the tional UNC93B1, showed a decreased number of activated incidence of death mainly attributed to infections was 3.0% exudate macrophages and the decreased expression of CXC vs. 8.3%, respectively (Fig. 2A). Evidence supporting this Chemokine Ligand (CXCL) 10, IFN-γ, and type I IFN in hypothesis may be seen in a previous report [26], in which the early phase of influenza A H1N1 infection. Given these two unrelated children possessing autosomal recessive previous findings, it is reasonable to assume from the pre- deficiency of UNC93B1 developed encephalitis due to sent study that the donor UNC93B1 C/C genotype, poten- herpes-simplex virus, wherein the antiviral cellular tially having higher inducibility of UNC93B1 than other responses were impaired in the interferon-α (IFN-α), IFN-β, genotypes, may enhance the functions of TLRs, thereby and IFN-λ pathways. Experimental studies [26–32], have augmenting infectious immunity, which improved the sur- shown that a lack or suppression of UNC93B1 inhibits the vival outcomes after allogeneic SCT. activation of TLR3, TLR7, and TLR9 to induce inflam- There is a contrasting report that the UNC93B1 mutation matory mediators, leading to increased susceptibility to with a 54-amino acid deletion in exon 4 was associated with infections. In addition, mouse model studies using the increased cardiac expression of IFN-β and markers of UNC93B1 mutant mice, which lack the UNC93B1 function tissue injury and fibrosis early after coxsackievirus strain B as a result of H412R missense mutation, exhibited increased serotype 3 (CVB3), which is a picornavirus that induces susceptibility to Toxoplasma gondii [27], Trypanosoma myocarditis [33]. However, one or more alternative path- cruzi [28], Leishmania major [29], and cytomegalovirus ways may mediate the cardiac IFN-β upregulation in the (CMV) [31] infections in association with the reduced case of CSV3. For example, melanoma differentiation- expression of inflammatory mediators, including IFN-γ associated gene 5 (MDA5) detects the double-stranded (T. cruzi, CMV, L. major), IFN-α (CMV) and interleukin RNA replicative form in picornavirus-infected cells and oo N-3HmlgB1 Homolog UNC-93 Donor Table 3 Multivariate analysis of the association between UNC93B1 polymorphism and post HSCT outcomes. Variable OS PFS TRM Relapse Adjusted HR 95% CI P Adjusted HR 95% CI P Adjusted HR 95% CI P Adjusted HR 95% CI P

Donor UNC93B1 rs308328 genotype, C/C vs. C/T or T/T 0.37 0.16–0.88 0 0.50 0.94–4.20 0.07 0.43 0.77–6.80 0.1 0.77 0.45–3.70 0.64 Recipient UNC93B1 rs308328 genotype, C/C vs. C/ 1.50 0.80–2.80 0.2 1.67 0.35–1.10 0.1 1.00 0.37–2.70 1.00 2.00 0.21–1.30 0.2 T or T/T ABO major mismatch 0.91 0.50–1.60 0.8 1.03 0.55–1.70 0.9 1.35 0.32–1.70 39 0.49 0.67 0.65–3.40 0.3 . . marrow. bone unrelated after outcomes survival predicts polymorphism genetic ABO minor mismatch 0.81 0.42–1.60 0.5 1.15 0.47–1.60 0.7 1.28 0.32–1.90 0.6 0.83 0.47–3.10 0.70 ABO bidirectional 0.70 0.25–2.00 0.5 1.32 0.27–2.10 0.60 1.52 0.14–3.10 0.60 0.71 0.31–6.00 0.7 Recipient age 1.00 1.00–1.00 <0.01 1.00 1.00–1.00 <0.01 1.00 1.00–1.10 0 1.00 0.98–1.00 0.9 Donor age 1.00 0.96–1.00 0.9 1.00 0.97–1.00 0.80 1.00 0.96–1.00 1 1.00 0.96–1.00 1 TNC 0.94 0.73–1.20 0.7 1.12 0.70–1.10 0.39 1.22 0.58–1.20 0.3 0.83 0.86–1.60 0.33 Pretransplantation CMV serostatus CMV-positive recipient 1.00 0.55–2.00 0.9 0.91 0.57–1.90 0.9 1.16 0.39–1.90 0.7 0.71 0.53–3.90 0.47 Missing 1.80 0.63–5.40 0.3 0.59 0.63–4.70 0.3 0.50 0.58–7.00 0.3 0.91 0.15–8.10 0.92 Disease stage standard risk/high risk 2.70 1.60–4.80 <0.01 0.34 1.70–5.00 <0.01 0.32 1.60–6.30 <0.01 0.67 0.56–3.90 0.43 Myeloid malignancies 1.40 0.79–2.50 0.3 0.67 0.88–2.60 0.1 0.67 0.70–3.30 0.28 0.91 0.40–2.80 0.90 Conditioning regimen, MAC vs. RIC 0.86 0.49–1.50 0.60 1.05 0.56–1.60 0.9 1.14 0.45–1.70 0.71 1.05 0.38–2.40 0.91 PS2–4 2.40 0.98–5.70 0.1 0.45 0.95–5.30 0.1 0.63 0.44–5.30 0.47 0.59 0.37–7.70 0.5 Year of HSCT 1.30 0.69–2.30 0.5 0.67 0.85–2.60 0.2 0.83 0.60–2.40 0.60 0.71 0.58–3.60 0.4 Recipient/donor sex match Female/male 0.79 0.46–1.40 0.4 1.14 0.53–1.50 0.6 1.09 0.48–1.80 0.80 1.11 0.37–2.20 0.8 Male/female 1.50 0.84–2.70 0.2 0.63 0.93–2.80 0.1 0.83 0.59–2.70 0.6 0.50 0.83–5.00 0.1 HSCT hematopoietic stem cell transplantation, HR hazard ratio, CI conﬁdence interval, OS overall survival, PFS progression-free survival, TRM transplant-related mortality, TNC total number of nucleated cells harvested, CMV cytomegalovirus, MAC myeloablative conditioning, RIC reduced-intensity conditioning, PS performance status. 40 K. Uchino et al.

appropriateness, was not obtained in the current study. Sec- ond, the functional roles of the SNP of UNC93B1 rs308328 in BMT remain purely speculative due to the lack of data using blood samples from BMT recipients and their donors. Further studies including more samples from patients, donors, and healthy individuals need to be conducted. Another limitation is that our results for the UNC93B1 gene may be a coincidence in the analysis of polymorphisms in many genes associated with the UNC93B1 gene, especially other TLR-related genes, although the fact that UNC93B1 is an important protein for infectious immunity that integrates the functions of TLRs and that the UNC93B1 gene plays a functional role suggests that the UNC93B1 gene polymorphism is probably responsible for the observed phenotype. Therefore, further studies using a separate cohort should be warranted to clarify whether polymorphisms in TLR-related genes are involved in the outcomes of BMT in an integrated manner. In conclusion, the findings of the present study sug- Fig. 2 The main causes of death were analyzed according to the gested that the donor UNC93B1 rs308328 C/C genotype UNC93B1 rs308328 genotype. ARDS acute respiratory distress predicted better survival outcomes after SCT than other syndrome, TMA thrombotic microangiopathy, IP interstitial pneumo- nia, GVHD graft-versus-host disease. genotypes. Therefore, the donor UNC93B1 rs308328 C/C genotype in donors may be a valuable tool for selecting donors and evaluating pretransplantation risks that, com- induces the production of inflammatory cytokines, includ- bined with other currently known risk factors, can form the ing IFN-β [30]. Since different immune system cascades are basis for carrying out suitable tailoring of transplantation activated depending on the type of pathogen, another cohort strategies. Considering the plausible functional roles of study to validate the current findings is desired in order to these polymorphisms, they may be candidates for future confirm the impact of UNC93B1 on allogeneic SCT, con- prophylactic and therapeutic strategies for complications sidering the type of pathogen, as this is beyond the scope of after allogeneic SCT and may lead to the development of the present study. molecular targeted therapy [39]. Further studies are war- Some reports have suggested that TLR4 polymorphisms ranted to ascertain whether or not the findings of this study may have an impact directly on the infection and graft can be extended to other stem cell sources or to HLA- rejection after transplantation [34–38]. We showed that the mismatched transplantation and to validate the present donor TLR4 + 3725G/G genotype, which induces a low findings in other ethnic groups. expression of TLR4, was significantly associated with a lower incidence of fatal infections than the donor G/C and C/C genotypes in another cohort of 367 patients who Materials and methods underwent unrelated HLA-matched BMT for hematologic malignancies through the Japan Marrow Donor Program Patients (JMDP) [22]. The UNC93B1 gene and the TLR4 gene are not predicted to be in linkage disequilibrium because A total of 237 patients and their unrelated donors were the UNC93B1 gene is located on chromosome 11q13, while included in the study. The patients underwent HLA- the TLR4 gene is located on chromosome 9q32-q33. Fur- matched BMT for hematologic malignancies through the thermore, the UNC93B1 gene does not affect the function of JMDP with T cell-replete marrow from HLA-A, HLA-B, TLR directly. Therefore, it is reasonable to consider that the HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 allele- donor UNC93B1 rs308328 C/C genotype, which putatively matched donors between May 2006 and April 2009. No has higher inducibility of UNC93B1 than others, improves patient had a history of any previous transplantation. The the outcomes of transplantation without the effects of final clinical data analyses of these patients were completed TLR4 SNPs. by September 27, 2011. Several limitations associated with the present study The diagnosed diseases were acute myeloid leukemia warrant mention. First, detailed information on the infections, (AML) (n = 115; 49%), acute lymphoblastic leukemia including the types, severity, treatments, and therapeutic (ALL) (n = 46; 19%), myelodysplastic syndrome (MDS) Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow. . . 41

(n = 38; 16%), malignant lymphoma (ML) (n = 24; 10%), infections, toxicities, and other non-relapse- or disease chronic myeloid leukemia (CML) (n = 10; 4%), myelopro- progression-related causes of death. The PFS was defined as liferative neoplasm (MPN) (n = 3; 1%), and multiple mye- the survival without disease relapse or progression. Any loma (MM) (n = 1; 0.4%) (Table 1). Relapse or secondary patients who were alive at the last follow-up date were cases were defined as high-risk diseases. Myeloid malig- censored. Data concerning the clinical and microbiologic nancies included AML, MDS, CML, and MPD. Lymphoid features of infections, postmortem changes, prophylaxis of malignancies included ALL, ML, and MM. infections, and therapy of GVHD given on an institutional The conditioning regimen depends on the type of under- basis were not available for this study. lying disease and the patient’s condition. The combination of The EZR software package [45] was used in all statistical cyclophosphamide (CY) and total body irradiation (TBI) was analyses. The probabilities of the OS and PFS were calcu- mainly classified as myeloablative conditioning (MAC), lated with the Kaplan–Meier method, and comparisons whereas the combination of fludarabine and melphalan was between groups were made with the log-rank test. The mainly classified as reduced-intensity conditioning (RIC) occurrence of TRM, disease relapse, aGVHD, and cGVHD [40]. Cyclosporin- or tacrolimus-based therapy was selected was compared using the Gray test [46] and analyzed using a as GVHD prophylaxis [41, 42]. Patients who were used anti- cumulative incidence analysis [47], considering relapse, T cell therapy, such as anti-thymocyte globulin or ex vivo death without disease relapse, death without aGVHD, T cell depletion were excluded from this study. death without cGVHD, and death without engraftment as All patients and donors provided their informed consent respective competing risks. A multivariate Cox model was at the time of transplantation to participate in molecular created for the OS, TRM, relapse, grades II–IV aGVHD, studies of this nature according to the Declaration of Hel- grades III–IV aGVHD, and cGVHD, using stepwise selec- sinki. This project was approved by the Institutional Review tion at a significance level of 5% to evaluate the hazard ratio Board of Aichi Medical University School of Medicine and (HR) associated with the UNC93B1 rs308328 genotype. the JMDP. The recipient age at the time of BMT, sex, pretransplantation CMV serostatus, performance status, disease char- UNC93B1 genotyping acteristics (i.e., disease type, disease lineage, and disease risk at transplantation), donor characteristics (i.e., age, sex, Real-time polymerase chain reaction (PCR) genotyping for sex compatibility, and ABO compatibility), transplant UNC93B1 was done with the TaqMan-Allelic discrimina- characteristics (i.e., MAC or RIC and total nucleated cell tion method in a Step One Plus Real-Time PCR system count harvested per recipient weight), and year of trans- (Applied Biosystems, Foster City, CA, USA) as described plantation were included as variables. The median was used previously [7], and the results were analyzed using the as the cut-off point for continuous variables. Allelic Discrimination software program (Applied Biosys- The chi-square test and Mann–Whitney test were tems). We purchased the specific probe designed for SNP used to compare the results of the two groups. The rs308328 (T>C) (product No. C_2623961_1) and TaqMan Hardy–Weinberg equilibrium for the UNC93B1 rs308328 genotyping master mix from Applied Biosystems. gene polymorphism was tested using the Haploview program. For both the univariate and multivariate analyses P < Data management and statistical analyses 0.05 was considered to indicate statistical significance.

The JMDP collected the data using a standardized report Acknowledgements This study was supported by grants from the form. Follow-up reports were submitted at 100 days, 1 year, Ministry of Education, Culture, Sports and Technology of Japan, a Research on Allergic Disease and Immunology (H26-106) in Health and annually after transplantation. Pre-transplant cytome- and Labor Science Grant from the Ministry of Health, Labour and galovirus (CMV) serostatus was routinely measured only in Welfare of Japan, the SENSHIN Medical Research Foundation patients but not in their donors. Neutrophil engraftment was (Osaka, Japan), the Aichi Cancer Research Foundation (Nagoya, confirmed by an absolute neutrophil count of more than Japan), and the 24th General Assembly of the Japanese Association of 9 Medical Sciences (Nagoya, Japan). The funders played no role in the 0.5 × 10 /L for at least three consecutive days. Acute study design, data collection, and analysis, the decision to publish, or GVHD (aGVHD) was diagnosed and graded in accordance the preparation of the manuscript. We thank all of the Japan Marrow with the established criteria [43]. Chronic GVHD (cGVHD) Donor Program (JMDP) transplant teams who provided valuable was classified on the basis of the Seattle criteria [44]. assistance in caring for the patients and donors evaluated in this study. assistance in caring for the patients and donors investigated in We calculated the OS from the date of transplantation to this study. death, regardless of cause, and defined disease relapse as the number of days from transplantation to disease relapse or Compliance with ethical standards progression. The TRM was defined as death due to any cause without relapse or disease progression, including Conflict of interest The authors declare no competing interests. 42 K. Uchino et al.

’ Publisher s note Springer Nature remains neutral with regard to 15. Schnetzke U, Spies-Weisshart B, Yomade O, Fischer M, Rachow T, fi jurisdictional claims in published maps and institutional af liations. Schrenk K, et al. Polymorphisms of Toll-like receptors (TLR2 and TLR4) are associated with the risk of infectious complications in acute myeloid leukemia. Genes Immun. 2015;16:83–8. References 16. Trejo-de la OA, Hernandez-Sancen P, Maldonado-Bernal C. Relevance of single-nucleotide polymorphisms in human TLR genes to infectious and inflammatory diseases and cancer. Genes 1. Gratwohl A, Brand R, Frassoni F, Rocha V, Niederwieser D, Immun. 2014;15:199–209. Reusser P, et al. Cause of death after allogeneic haematopoietic 17. Shey MS, Randhawa AK, Bowmaker M, Smith E, Scriba TJ, de stem cell transplantation (HSCT) in early leukaemias: an EBMT Kock M, et al. Single nucleotide polymorphisms in toll-like receptor analysis of lethal infectious complications and changes over 6 are associated with altered lipopeptide- and mycobacteria-induced – calendar time. Bone Marrow Transpl. 2005;36:757 69. interleukin-6 secretion. Genes Immun. 2010;11:561–72. 2. Nomoto H, Takami A, Espinoza JL, Onizuka M, Kashiwase K, 18. Heidegger S, van den Brink MR, Haas T, Poeck H. The role of Morishima Y, et al. Recipient ADAMTS13 single-nucleotide pattern-recognition receptors in graft-versus-host disease and polymorphism predicts relapse after unrelated bone marrow trans- graft-versus-leukemia after allogeneic stem cell transplantation. plantation for hematologic malignancy. Int J Mol Sci. 2019;20:214. Front Immunol. 2014;5:337. 3. Takami A, Espinoza JL, Onizuka M, Ishiyama K, Kawase T, 19. Penack O, Holler E, van den Brink MR. Graft-versus-host disease: Kanda Y, et al. A single-nucleotide polymorphism of the regulation by microbe-associated molecules and innate immune Fcgamma receptor type IIIA gene in the recipient predicts trans- receptors. Blood. 2010;115:1865–72. plant outcomes after HLA fully matched unrelated BMT for 20. Maeda Y. Pathogenesis of graft-versus-host disease: innate – myeloid malignancies. Bone Marrow Transpl. 2011;46:238 43. immunity amplifying acute alloimmune responses. Int J Hematol. 4. Espinoza JL, Takami A, Onizuka M, Morishima Y, Fukuda T, 2013;98:293–9. Kodera Y, et al. Recipient PTPN22 -1123 C/C genotype predicts 21. Tu S, Zhong D, Xie W, Huang W, Jiang Y, Li Y. Role of toll-like acute graft-versus-host disease after HLA fully matched unrelated receptor signaling in the pathogenesis of graft-versus-host dis- bone marrow transplantation for hematologic malignancies. Biol eases. Int J Mol Sci. 2016;17:1288. – Blood Marrow Transplant. 2013;19:240 6. 22. Uchino K, Mizuno S, Sato-Otsubo A, Nannya Y, Mizutani M, 5. Nakata K, Takami A, Espinoza JL, Matsuo K, Morishima Y, Horio T, et al. Toll-like receptor genetic variations in bone mar- + Onizuka M, et al. The recipient CXCL10 1642C>G variation row transplantation. Oncotarget. 2017;8:45670–86. predicts survival outcomes after HLA fully matched unrelated 23. Uchino K, Mizuno S, Mizutani M, Horio T, Hanamura I, Espinoza JL, – bone marrow transplantation. Clin Immunol. 2013;146:104 11. et al. Toll-like receptor 1 variation increases the risk of transplant- 6. Horio T, Mizuno S, Uchino K, Mizutani M, Hanamura I, Espinoza related mortality in hematologic malignancies. Transpl Immunol. JL, et al. The recipient CCR5 variation predicts survival outcomes 2016;38:60–6. – after bone marrow transplantation. Transpl Immunol. 2017;42:34 9. 24. Kim YM, Brinkmann MM, Paquet ME, Ploegh HL. UNC93B1 7. Espinoza JL, Takami A, Onizuka M, Sao H, Akiyama H, Miya- delivers nucleotide-sensing toll-like receptors to endolysosomes. fi mura K, et al. NKG2D gene polymorphism has a signi cant Nature. 2008;452:234–8. impact on transplant outcomes after HLA-fully-matched unrelated 25. Inoue N, Katsumata Y, Watanabe M, Ishido N, Manabe Y, Wata- bone marrow transplantation for standard risk hematologic nabe A, et al. Polymorphisms and expression of toll-like receptors in – malignancies. Haematologica. 2009;94:1427 34. autoimmune thyroid diseases. Autoimmunity. 2017;50:182–91. 8. Espinoza LJ, Takami A, Nakata K, Yamada K, Onizuka M, 26. Casrouge A, Zhang SY, Eidenschenk C, Jouanguy E, Puel A, Kawase T, et al. Genetic variants of human granzyme B predict Yang K, et al. Herpes simplex virus encephalitis in human UNC- transplant outcomes after HLA matched unrelated bone marrow 93B deficiency. Science. 2006;314:308–12. transplantation for myeloid malignancies. PLoS ONE. 2011;6: 27. Melo MB, Kasperkovitz P, Cerny A, Konen-Waisman S, Kurt- e23827. Jones EA, Lien E, et al. UNC93B1 mediates host resistance to 9. Espinoza JL, Takami A, Nakata K, Onizuka M, Kawase T, infection with Toxoplasma gondii. PLoS Pathog. 2010;6:e1001071. Akiyama H, et al. A genetic variant in the IL-17 promoter is 28. Caetano BC, Carmo BB, Melo MB, Cerny A, dos Santos SL, functionally associated with acute graft-versus-host disease after Bartholomeu DC, et al. Requirement of UNC93B1 reveals a cri- unrelated bone marrow transplantation. PLoS ONE. 2011;6:e26229. tical role for TLR7 in host resistance to primary infection with 10. Nomoto H, Takami A, Espinoza JL, Matsuo K, Mizuno S, Oni- Trypanosoma cruzi. J Immunol. 2011;187:1903–11. zuka M, et al. A donor thrombomodulin gene variation predicts 29. Schamber-Reis BLF, Petritus PM, Caetano BC, Martinez ER, graft-versus-host disease development and mortality after bone Okuda K, Golenbock D, et al. UNC93B1 and nucleic acid-sensing – marrow transplantation. Int J Hematol. 2015;102:460 70. toll-like receptors mediate host resistance to infection with 11. Suetsugu K, Mori Y, Yamamoto N, Shigematsu T, Miyamoto T, Leishmania major. J Biol Chem. 2013;288:7127–36. Egashira N, et al. Impact of CYP3A5, POR, and CYP2C19 30. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, polymorphisms on trough concentration to dose ratio of tacroli- et al. Differential roles of MDA5 and RIG-I helicases in the recog- mus in allogeneic hematopoietic stem cell transplantation. Int J nition of RNA viruses. Nature. 2006;441:101–5. Mol Sci. 2019;20:2413. 31. Crane MJ, Gaddi PJ, Salazar-Mather TP. UNC93B1 mediates 12. Koldej RM, Perera T, Collins J, Ritchie DS. Association between innate inflammation and antiviral defense in the liver during acute P2X7 polymorphisms and post-transplant outcomes in allogeneic murine cytomegalovirus infection. PLoS ONE. 2012;7:e39161. haematopoietic stem cell transplantation. Int J Mol Sci. 32. Lafferty EI, Flaczyk A, Angers I, Homer R, d’Hennezel E, Malo D, 2020;21:3772. et al. An ENU-induced splicing mutation reveals a role for 13. Horio T, Morishita E, Mizuno S, Uchino K, Hanamura I, Espinoza Unc93b1 in early immune cell activation following influenza A JL, et al. Donor heme oxygenase-1 promoter gene polymorphism H1N1 infection. Genes Immun. 2014;15:320–32. predicts survival after unrelated bone marrow transplantation for 33. Lafferty EI, Wiltshire SA, Angers I, Vidal SM, Qureshi ST. high-risk patients. Cancers 2020;12:424. Unc93b1-dependent endosomal toll-like receptor signaling reg- fl 14. Takeuchi O, Akira S. Pattern recognition receptors and in am- ulates inflammation and mortality during coxsackievirus B3 – mation. Cell. 2010;140:805 20. infection. J Innate Immun. 2015;7:315–30. Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow. . . 43

34. Noreen M, Shah MA, Mall SM, Choudhary S, Hussain T, Ahmed I, 41. Nash RA, Antin JH, Karanes C, Fay JW, Avalos BR, Yeager AM, et al. TLR4 polymorphisms and disease susceptibility. Inflamm et al. Phase 3 study comparing methotrexate and tacrolimus with Res. 2012;61:177–88. methotrexate and cyclosporine for prophylaxis of acute graft- 35. Oetting WS, Guan W, Schladt DP, Leduc RE, Jacobson PA, versus-host disease after marrow transplantation from unrelated Matas AJ, et al. Donor polymorphisms of toll-like receptor 4 donors. Blood. 2000;96:2062–8. associated with graft failure in liver transplant recipients. Liver 42. Storb R, Deeg HJ, Whitehead J, Appelbaum F, Beatty P, Ben- Transpl. 2012;18:1399–405. singer W, et al. Methotrexate and cyclosporine compared with 36. Lee SO, Brown RA, Kang SH, Abdel Massih RC, Razonable RR. cyclosporine alone for prophylaxis of acute graft versus host Toll-like receptor 4 polymorphisms and the risk of gram-negative disease after marrow transplantation for leukemia. N Engl J Med. bacterial infections after liver transplantation. Transplantation. 1986;314:729–35. 2011;92:690–6. 43. Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, 37. Dhillon N, Walsh L, Kruger B, Ward SC, Godbold JH, Radwan M, Hows J, et al. 1994 Consensus conference on acute GVHD et al. A single nucleotide polymorphism of Toll-like receptor 4 grading. Bone Marrow Transpl. 1995;15:825–8. identifies the risk of developing graft failure after liver transplan- 44. Shulman HM, Sullivan KM, Weiden PL, McDonald GB, Striker tation. J Hepatol. 2010;53:67–72. GE, Sale GE, et al. Chronic graft-versus-host syndrome in man. A 38. Hwang YH, Ro H, Choi I, Kim H, Oh KH, Hwang JI, et al. Impact long-term clinicopathologic study of 20 Seattle patients. Am J of polymorphisms of TLR4/CD14 and TLR3 on acute rejection in Med. 1980;69:204–17. kidney transplantation. Transplantation. 2009;88:699–705. 45. Kanda Y. Investigation of the freely available easy-to-use software 39. Raschella G, Melino G, Gambacurta A. Cell death in cancer in the ‘EZR’ for medical statistics. Bone Marrow Transpl. 2013;48:452–8. era of precision medicine. Genes Immun. 2019;20:529–38. 46. Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of 40. Giralt S, Ballen K, Rizzo D, Bacigalupo A, Horowitz M, Pasquini M, failure probabilities in the presence of competing risks: new et al. Reduced-intensity conditioning regimen workshop: defining the representations of old estimators. Stat Med. 1999;18:695–706. dose spectrum. Report of a workshop convened by the center for 47. Scrucca L, Santucci A, Aversa F. Competing risk analysis international blood and marrow transplant research. Biol Blood using R: an easy guide for clinicians. Bone Marrow Transpl. Marrow Transplant. 2009;15:367–9. 2007;40:381–7.